Methods for shp1 mediated neuroprotection

ABSTRACT

The effect of EPO on the phosphorylation of the EPO receptor, the activation of the MAP Kinase pathway, and the expression of SHP-1 were analyzed. EPO was observed to cause a decrease in the expression of its negative regulator SHP-1. The decrease observed at both the mRNA and protein level was dose dependent and persisted as long as 24 hr following EPO treatment. EPO can down regulate the expression of its own negative regulator as a means for increased potency in neurons. Assays were generated to identify compounds that are useful in regulating SHP1 activity in neural cells.

This application is a divisional application and claims priority fromU.S. application Ser. No. 10/386,243, filed Mar. 11, 2003. The completedisclosure of the aforementioned U.S. patent application is herebyincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides methods to use SHP1 modulators forneuroprotection and assays capable of identifying SHP1 modulators.

BACKGROUND OF THE INVENTION

This patent application claims priority from U.S. Provisional patentapplication No. 60/363,440 filed Mar. 11, 2002 and entitled “Methods andCompositions for SHP1 mediated Neuroprotection.”

Erythropoietin (EPO) is responsible for the maintenance, increase andterminal differentiation of erythroid progenitor cells. Erythropoiesisis controlled by the production of erythropoictin in the kidney as afunction of oxygen tension in the blood. Recent data has shown thatother cell types, including endothelial cells [26], kidney cells [33]and neuronal cells [22], also contain functional EPO receptor (EPOR).

Several lines of evidence support the function of EPO as aneuroprotective molecule. First, both EPO and its receptor have beenfound in neural tissues [11], including rat hippocampal neurons andsubsets of neurons in the rat cerebral cortex [21]. Second, in a seriesof in vitro experiments, EPO has been shown to prevent the death ofneurons in response to a variety of insults including glutamate toxicityand hypoxia [21, 28]. In addition, the neuroprotective effects of EPOhave been demonstrated in several in vivo models of CNS injury.Intraventricular administration of EPO significantly decreased thedamage observed in an ischemic gerbil model [27], and in a mouse modelof middle cerebral a occlusion [3]. Recently, reports have shown thatEPO administered systemically can cross the blood brain barrier and havea protective effect in in vivo models of stroke blunt trauma [5] and suarachnoid hemorrhage-induced acute cerebral ischemia [1]. In addition,EPO has been suggested to be the factor responsible for the increasedsurvival, differentiation and proliferation of neuronal stem cells thatare cultured under conditions of low oxygen [30]. These result suggestthat EPO can be useful as a neuroprotective molecule for the treatmentof nervous system conditions, such as stroke, and otherneurodegenerative diseases.

U.S. Pat. No. 6,165,783 states that “[t]he erythropoietin may beexogenously applied to the multipotent neural stem cells, oralternatively, the cells can be subjected to hypoxic insult whichinduces the cells to express erythropoietin.”

EPO exerts its biological effect by binding to preexisting receptordimers located on the cell and inducing a conformational change in theEPO receptor, EPOR. The change in EPOR conformation results in theactivation of JAK2 and the subsequent phosphorylation of tyrosineresidues on the EPO receptor and other intracellular proteins such asERKs, She and Stat5 [6, 4]. The change in EPOR conformation also resultsin recruitments of SH2 domain containing proteins to the receptorcomplex, including the SH2 containing protein tyrosine phosphatase(PTPase) SHP1, also known as SH-PTP1, PTP1C, HCP or SHP, whichdephosphorylates JAK2 and subsequently inactivates JAK2. Due to thepresence of high levels of SHP1 in all hematopoietic cell lineages atall stages of hematopoietic cell differentiation, phosphorylation of EPOis transient with the loss of detectable phosphorylation on tyrosineresidue(s) of EPOR within thin minutes after the initial binding of EPOto EPOR. While the function of EPO to protect neurons from cell death isconsistent with the function of EPO in hematopoietic cells, the potencyof the effect of EPO on neuronal cells increased significantly ascompared with hematopoietic cells. EPO has been observed to be effectiveat pM concentrations in neurons while nM levels of EPO are required toelicit the same effects in hematopoietic cells [27, 13].

The mechanism for increased potency of EPO in neuronal cells is notclear. Previous reports have indicate, that the EPOR expressed inneurons is identical in sequence to that found in hematopoietic cells,including all potential sites of tyrosine phosphorylation, and that EPORexpressed in neurons has a lower affinity for EPO [18]. While thepossibility exists that the EPOR is different in neurons due tovariations in post-translational modifications, the relative lowaffinity of EPO for the EPOR in neurons and the observation that EPOactivates similar signaling pathways to neuronal cells similar to thosein hematopoietic cells make it unlikely that changes in EPOR aloneaccounts for the increased potency of EPO in neuronal cells.

Little is known about the role of SHP1 in EPOR activated signaltransduction pathway in cells of the nervous system in part becausethere is little information concerning the expression or function ofSHP1 in CNS. SHP1 has been reported to be present in specific neuronsubtypes [10] and to negatively regulate specific functions in rob celllines [20]. Stimulation of neutrophils with chemotactic peptides isknown to result in the activation of tyrosine kinases that mediateneutrophil responses (Cui et al., J. Immunol., 1994) and the PTPaseactivity of SHP1 modulates agonist-induced activity by reversing theeffects of tyrosine kinases activated in the initial phases of cellstimulation U.S. Pat. No. 6,261,279 suggests that “agents that couldstimulate PTPase activity could have potential therapeutic applicationsas anti-inflammatory mediators.” Although it has been reported that SHP1can act as a positive signal in RAS-mediated activation of themitogen-activated protein kinase pathway, it is not known whatregulatory role SHP1 plays in EPOR activated signal transduction pathwayin cells of the nervous system.

Understanding the role of SHP1 in EPOR activated signal transductionpathway in cells of the nervous system can shed light on the mechanismof the neuroprotection function of EPO, which in turn can help thedesign and identification of novel neuroprotective molecules.

SUMMARY OF INVENTION

It has now been discovered that treating cells of the nervous systemwith EPO results in decreased expression of SHP1 in the cell. This downregulation of SHP1 then prevents the timely dephosphorylation of theEPOR thus allowing for a sustained activation of both the EPOR itselfand downstream targets such as ERK1/ERK2. The sustained activation ofEPOR can explain the high potency of EPO in the cells of nervous system.

In one general aspect, the invention therefore provides a method fortreating a nervous system condition related to EPOR in a subject in needthereof, comprising the step of administering to the subject atherapeutically effective dose of a composition that decrease thetyrosine phosphatase activity of a SHP1 in a cell of the nerve system ofthe subject. For example, the subject can be in need of treatment forneuroprotection.

In other general aspects, the invention provides a method for tang anervous system on related to EPOR in a subject in need thereof,comprising the step of administering to the subject a therapeuticallyeffective dose of a composition that decreases the expression of a SHP1in a cell of the nerve system of the subject. For example, thecomposition comprises an antisense nucleic acid or a siRNA moleculespecific for an SHP1 gene and the antisense nucleic acid or siRNAmolecule specifically suppresses SHP1 gene expression.

An additional general aspect of the invention is a method of identifyinga compound useful for treating a nervous system condition related toEPOR, comprising the steps of:

1) contacting a test compound with an SHP1 protein or an active fragmentthereof; and

2) determining the ability of the test compound to decease the tyrosinephosphatase activity of SHP1.

Further general aspect of the invention is a method of identifying acompound useful for treating a nervous system condition related to EPOR,comprising the steps of:

-   -   a) contacting a test compound with a regulatory sequence for a        SHP1 gene or a cellular component that binds to the regulatory        sequence for a SHP1 gene; and    -   b) determining whether the test compound decreases the        expression of a gene controlled by said regulatory sequence.

Yet another general aspect of the invention is a method of identifying acompound useful for treating a nervous system condition related to EPOR,comprising the steps of:

-   -   a) combining a test compound, a labeled ligand for a SHP1        protein, and a SHP1 protein or an active fragment thereof; and    -   b) measuring the binding of the test compound to the SHP1        protein or active fragment thereof by a reduction in the amount        of labeled ligand binding to the SHP1 protein or active fragment        thereof.

In preferred embodiments of the invention, the methods of identifying acompound useful for treating a nervous system condition related to EPOR,further comprise the steps of

-   -   a) contacting a neuronal cell with the test compound;    -   b) inducing neurotoxicity in the neuronal cell;    -   c) assaying the cell survival rate in the presence of the test        compound,    -   and comparing the cell survival rate with that of a control        wherein the neuronal cell is not treated with the test compound.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiment and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Illustrates a sustained phosphorylation of ERKs in neuronstreated with EPO as compared to that in hematopoietic cells treated withEPO. Primary cortical neurons and hematopoietic cells UT-7 were treatedwith EPO for the indicated time then harvested. Cell extracts wereanalyzed by SDS-PAGE Western blots, probed with antibodies againstERK1/ERK2 (p44/42) MAPK. The time course of ERK1/ERK2 phosphorylation inneurons or UT-7 cells treated with EPO is plotted.

FIG. 2: Illustrates a sustained phosphorylation of EPOR in neuronstreated with EPO as compared to that in hematopoietic cells treated withEPO. Primary cortical neurons and hematopoietic cells UT-7 were treatedwith EPO for the indicated time then harvested. Cell extractsimmunoprecipitated with anti-EPOR antibody and blotted with either ananti-EPOR antibody or an anti-phosphotyrosine antibody. The time courseof EPOR phosphorylation in neurons or UT-7 cells d with EPO was plotted.

FIG. 3: Demostats that following treatment with EPO, SHP1 mRNA levelsdecreased dose dependantly in neurons but maintained the same inhematopoietic cells. Primary cortical neurons and hematopoietic cellsUT-7 were left (1) untreated or treated with (2) 100 fM, (3) 10 pM or(4) 1 nM EPO for 24 hours. Total RNA was isolated and levels of mRNA forSHP1 and the housekeeping gene Glyceraldehye-3-phosphate dehydrogenase(GAPDH) were determined using RT-PCR. SHP1 mRNA levels showed a dosedependant decrease following treatment with EPO in neurons. SHP1 mRNAlevels were unchanged in EPO treated UT-7 cells.

FIG. 4: Shows that following treatment with EPO for time periods lastingfrom 3 hours to 24 hours, SHP1 mRNA levels continued to decrease inneurons while remained the same in hematopoietic cells. Primary corticalneurons and hematopoietic cells UT-7 were simulated with EPO (2.5 nM and10 pM respectively) for 3, 6, or 24 hours. Total RNA was subjected toRT-PCR to determine mRNA levels of SHP1 and GAPDH. EPO treatment causeda decrease in SHP1 mRNA levels by 3 hours and continued to out to 24hours. SHP1 mRNA levels in U-7 cells remained unchanged followingtreatment with EPO.

FIG. 5: Demonstrates that following treatment with EPO, SHP1 proteinlevels decreased with time in neurons, but maintained the same in UT-7cells. Primary cortical neurons and hematopoietic cells UT-7 werestimulated with EPO (2.5 nM and 10 pM respectively) for the indicatedtime points. Cell extracts were collected and subject to SDS-PAGE andimmobilized in a nylon membrane. Blots were then probed with and anantibody against SHP1. A decrease in the level of SHP1 protein wasdetectable at 3 hours and 6 hours in cortical neurons. No detectablechange in SHP1 levels was observed in UT-7 cells treated with EPO.

FIG. 6: Illustrates that EPO caused a dose dependant decrease in SHP1mRNA in non-neuronal nervous cell astrocytes with the most significantchanges at 10 pM and 1 nM. Primary astrocytes were (1) left untreated ortreated with (2) 100 fM, (3) 10 pM or (4) 1 nM EPO for 24 hours. TotalRNA was isolated and subjected to RT-PCR to determine levels of SHP1mRNA. EPO a dose dependant decrease in SHP1 mRNA in astrocytes with themost significant changes at 10 pM and 1 nM.

FIG. 7, Panels A and B:

An illusion of increased potency of EPO in neurons due to EPO downregulation of SHP1. In hematopoietic cells (5a), EPOR activation isinitiated by EPO binding to the receptor and the subsequent activationof JAK2 and phosphorylation of specific tyrosine residues located on theEPOR. Inactivation (termination) of EPOR signaling is achieved, at leastin a large part, by the removal of these phosphate groups by thetyrosine phosphatase SHP1. Thus, the balance between these two opposingmechanisms determines the amount of activated EPOR in response to aspecific concentration of EPO. In cells of the nervous system (5b),binding of EPO to EPOR causes a rapid down-regulation of the expressionof SHP1. The decrease in SHP-1 protein levels present in the cytosolresults in less dephosphorylation of EPOR, and shifts theactivation/inactivation balance toward activation Therefore at a givenconcentration of EPO, EPORs are more likely to be activated thaninactivated leading to an increased potency for EPO in these cells

DETAILED DESCRIPTION OF THE INVENTION

All publications cited below are hereby incorporated by reference.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains.

The following are abbreviations that are at times used in thisspecification below.

bp=base pair

cDNA=complementary DNA

C=central nervous system

EPO=erythropoietin

EPOR=erythropoietin receptor

MAP kinase=mitogen-activated protein kinase

PAGE=polyacrylamide gel electrophoresis

PCR=polymerase chain reaction

PTPase=protein tyrosine phosphatase

SDS=sodium dodecyl sulfate

SH2=src homology 2

SiRNA=small interfering RNA UTR=untranslated region

The terms “including,” “comprising”, “containing” and “having” are usedherein in their open, non-limiting sense.

An “activity”, a “biological activity”, or a “functional activity” of apolypeptide or nucleic acid of the invention refers to an activityexerted by a polypeptide or nucleic acid molecule of the invention asdetermined in vivo or in vitro, according to standard techniques. Suchactivities can be a direct activity, such as an association with or anenzymatic activity on a second protein, or an indirect activity, such asa cellular signaling activity mediated by interaction of the proteinwith a second protein.

An exemplary biological activity associated with EPOR is its ability tobind EPO. Another exemplary biological activity of EPOR is the abilityof EPOR, upon binding of EPO, to transduce a signal to the targetproteins such as JAK2, ERKs, Shc and Stat5. Yet, another exemplarbiological activity of EPOR is the ability of EPOR, upon binding of EPO,to recruit SH2 domain containing proteins, including SHP1, to thereceptor complex.

An exemplary activity of SHP1 is the tyrosine phosphatase activity ofSHP1, for example, the ability of SHP1 to dephosphorylate thephosphorylated JAK2. Another exemplary activity of SHP1 is the abilityof SHP1 to bind to a tyrosine residue in the cytoplasmic domain of EPORupon the binding of EPO to EPOR.

A “nervous system condition related to EPOR” shall include a disorder ordisease associated with insufficient activity of EPOR in the nervoussystem, and conditions that accompany this disorder or disease.“Insufficient activity of EPOR” refers to either 1) the absence of EPORexpression in cells which normally express EPOR; 2) deceased EPORexpression; 3) decreased activity of EPOR per unit of the EPOR protein;or 4) mutations leading to constitutive inactivation of one or more EPORbiological activities. Exemplary nervous system conditions related toEPOR, include, but are not limited to conditions that are the result ofa seizure disorder, multiple sclerosis, stoke, hypotension, cardiacarrest, ischemia, myocardial infarction, inflammation, aging orcognitive dysfunction, radiation damage, cerebral palsy,neurodegenerative disease, Alzheimer's disease, Parkinson's disease,Leigh disease, AIDS dementia, memory loss, amyotrophic lateralsclerosis, alcoholism, mood disorder, anxiety disorder, attentiondeficit disorder, autism, Creutzfeld-Jakob disease, brain or-spinal cordtrauma, heart-lung bypass, glaucoma, retinal ischemia, or retinaltrauma.

“Gene therapy” means the introduction of a functional gene, genes, or anucleic acid fragment from some source by any suitable means into aliving cell to correct for a genetic defect.

The term “regulatory region” or “regulatory sequence” is intended toinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals, and ribosome binding site (for bacterialexpression) and, an operator). Such regulatory sequences are describedand can be readily determined using a variety of methods known to thoseskilled in the art (see for example, in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic San Diego, Calif.(1990). Regulatory sequences include those at direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosethat direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences).

“Cells of the nervous system” refers to cells to cells that arespecifically related to the nervous system of an animal. For example, a“cell of the nervous system” can be a “neuron” or a “nerve cell”, whichis an excitable cell specialized for the transmission of electricalsignals over long distances. Neurons receive input from sensory cells orother neurons and send output to muscles or other neurons. Exemplary“neurons”, include a “sensory neuron” that has sensory input, a“motoneuron” that has muscle outputs, or “interneuron” that connectsonly with other neurons. A “cell of the nervous system” can also be aspecialized non-neuronal nervous cell, for example a glial cell, whichis a cell that surrounds a neuron, providing mechanical and physicalsupport and electrical insulation between neurons. Examples of glialcells include, but are not limited to, microglial cells and astrocytes.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who is the object of treatment,observation or experiment.

As used herein, the term “substantially purified” means that the proteinor biologically active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the protein is derived, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus,protein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of heterologous protein (also referred to herein as a“contaminating protein”). When the protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium resents lessthan about 20%, 10%, or 5% of the volume of the protein preparation.When the protein is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals that areinvolved in the synthesis of the protein. Accordingly such preparationsof the protein have less than about 30%, 20%, 10%, 5% (by dry weight) ofchemical precursors or compounds other than the polypeptide of interest.

As used herein, “treating” a disorder means eliminating or otherwiseameliorating the cause and/or effects thereof. To “inhibit” or“inhibiting” the onset of a disorder means preventing, delaying orreducing the likelihood of such onset.

The term “composition” is intended to encompass a product comprising thespecified ingredients in the specified amounts, as well as any productwhich results, directly or indirectly, from combinations of thespecified ingredients in the specified amounts.

As used herein, “erythropoietin” or “EPO” shall include thosepolypeptides and proteins that have the biological activity ofrecombinant human erythropoietin (rhEPO), as well as erythropoietinanalogs, erythropoietin isoforms, erythropoietin mimetics,erythropoietin fragments, hybrid erythropoietin proteins, fusionproteins oligomers and multimers of the above, homologues of the above,glycosylation pattern variants of the above, and muteins of the above,regardless of the biological activity of same, and further regardless ofthe method of synthesis or manufacture thereof including, but notlimited to, recombinant (whether produced from cDNA or genomic DNA),synthetic, transgenic, and gene activated methods. Specific examples oferythropoietin include, Epoetin alfa (EPREX®, ERYPO®, PROCRIT®), novelerythropoiesis stimulating protein (NESP™, ARANESP™, darbepoetin alfa)such as the hyperglycosylated analog of recombinant human erythropoictin(Epoetin) described in European patent application EP 640619, humanerythropoietin analog (such as the human serum albumin fusion proteinsdescribed in the international patent application WO 99/66054),erythropoietin mutants described in the international patent applicationWO 99/38890, erythropoietin omega, which may be produced from an Apa Irestriction fragment of the human erythropoietin gene described in U.S.Pat. No. 5,688,679, alter glycosylated human erythropoietin described inthe international patent application WO 99/11781 and EP 1064951, PEGconjugated erythropoietin analogs described in WO 98/05363 or U.S. Pat.No. 5,643,575. Specific examples of cell lines modified for expressionof endogenous human erythropoietin are described in international patentapplications WO 99/05268 and WO 94/12650. The generally preferred formof EPO is purified recombinant human EPO (rhEPO), currently formulatedand distributed under the trademarks of EPREX®, ERYPO®, PROCRIT® orARANESP™.

The term “SHP1”, “SH-PTP1”, “PTP1C”, “HCP” or “SHP” all refers to a srchomology 2-containing tyrosine phosphatase that is capable of removing aphosphate group from a phosphorylated protein at a tyrosine residue, andcomprises an amino acid sequence that has greater than about 60% aminoacid sequence identity, to human SHP1 (Plutzky et al., 1992. Proc. Natl,Acad. Sci U.S.A. 89 (3), 1123-1127, GenBank Protein_Id No.: AAA36610);is capable of binding to antibodies, e.g., polyclonal or monoclonalantibodies, raised against a human SHP1 protein described herein; or isencoded by a polynucleotide that specifically hybridizes under highstringent hybridization conditions to a nucleic acid molecule having asequence that has greater than about 80% nucleotide sequence identity tohuman SHP1 cDNA (GenBank nucleotide Accession No: M77273).

Exemplary high stringency or stringent hybridization conditions include:50% a formamide, 5×SSC and 1% SDS incubated at 42° C. or 5×SSC and 1%SDS incubated at 65° C., with a wash in 0.2×SSC and 0.1% SDS at 65° C.

In preferred embodiments, the SHP1 is a polypeptide having greater an80, 85, 90, or 95 percent amino acid sequence identity to human SHP1. Inother preferred embodiments, the SHP1 is a polypeptide encoded by apolynucleotide that specifically hybridizes under stringenthybridization conditions to a nucleic acid molecule having a sequencethat has greater than 80, 85, 90, or 95 percent nucleotide sequenceidentity to human SHP1 cDNA. Exemplary SHP1 include SHP1 or orthologsthat have been identified in human rat, mouse, and other animals,including pig and monkey.

An “active fragment of SHP1” refers to a fragment of SHP1 that comprisesan amino acid sequence that has greater than about 90% amino acidsequence identity, preferably about 95% amino acid sequence identity, tothe sequence of at least ten consecutive amino acids of a SHP1, and suchan active fragment of SHP1 is still capable of removing a phosphategroup from a phosphorylated protein at a tyrosine residue.

“Promoter” means a regulatory sequence of DNA that is involved in thebinding of RNA polymerase to initiate transcription of a gene. A “gene”is a segment of DNA involved in producing a peptide, polypeptide, orprotein, including the coding region, noncoding regions preceding(“5′UTR”) and following (“340 UTR”) coding region, as well asintervening non-coding sequences (“introns”) between individual codingsegments (“exons”). A promoter is herein considered as a part of thecorresponding gene. Coding refers to the representation of auto acid,start and stop signals in a three base “triplet” code. Promoters areoften upstream (“5′ to”) of the transcription initiation site of thegene.

Methods of Treament

In hematopoietic and nonhematopoietic cells, including neurons, SHP-1decreases the level of EPOR phosphorylation by binding to a tyrosineresidue in the cytoplasmic domain of the EPOR and dephosphorylatingJAK2, which is phosphorylated upon EPO binding to the EPOR [7]. Inhematopoietic cells, the phosphorylation of EPOR is transient largelydue to the presence of high levels of SHP1 activity inside the cell. Thepresent invention shows that although EPO utilizes similar signalingpathways in neurons and hematopoietic cells, the temporal signalingevents are different. EPO down-regulates SHP1 expression in cells ofnervous system resulting in a sustained phosphorylation of the EPOreceptor following EPO binding. Moreover, a sustained phosphorylation ofthe downstream signaling proteins ERK1/ERK2 is observed follow EPObinding and EPO receptor activation. These observations provide insistas to why EPO has increased potency in neurons.

In one general aspect, the invention provides a method for treating anervous system condition related to EPOR in a subject in need thereof,comprising the step of administering to the subject a therapeuticallyeffective dose of a composition that decreases the tyrosine phosphataseactivity of a SHP1 in a cell of the nervous system of a subject. In apreferred embodiment, the subject receiving treatment is in need ofneuroprotection. The neuroprotective effects of EPO are useful inindividuals suffering from seizure disorders, multiple sclerosis,stroke, hypotension, cardiac arrest, ischemia, myocardial infarction,inflammation, aging or cognitive dysfunction, radiation damage, cerebralpalsy, neurodegenerative disease, Alzheimer's disease, Pakinson'sdisease Leigh disease, AIDS dementia, memory loss, amyotrophic lateralsclerosis, alcoholism, mood disorder, anxiety disorder, attentiondeficit disorder, autism, Creutzfeld-Jakob disease, brain or-spinal cordtrauma, heat-lung bypass, glaucoma, retinal ischemia, or retinal trauma.In one preferred embodiment, the condition is an acute nervous systemdisease selected from the group consisting of ischemic stoke,hemorrhagic stroke, spinal cord injury traumatic brain injury, and thelike. In another preferred embodiment the condition is a chronic nervoussystem disease selected form the group consisting of Alzheimer'sdisease, Parkinson's disease, peripheral neuropathies, and cognitiveimpairment associated with coronary artery bypass graft surgery (CABG)and carotid endarterectomy (CEA).

In one preferred embodiment, the cell of the nervous system is aneuronal cell, such as a sensory neuron, a motoneuron, or interneuron.Particularly, the neon is a primary cortical neuron, or a hippocamapalneuron. In another preferred embodiment, the cell of the nervous systemis a non-neuronal cell, such as a glial cell. Particularly, thenon-neuronal cells of the nervous system are microglial cells orastrocytes.

The composition that decreases the tyrosine phosphatase activity of aSHP1 in a cell of the nervous system of the subject comprises a compoundthat is identified by any of the compound identification methodsdescribed infra. For example, the composition can comprise a compoundwith the structure of formula (I), formula (II), or formula (III) orother compositions identified using the assays of the pr invention anddescribed infra.

The composition can be administered in combination with one or moreneuroprotective agents. In one embodiment of this aspect, more man onecompound that decreases the tyrosine phosphatase activity of a SHP1 in acell of the nerve system of the subject can be administer to thesubject. In a preferred embodiment, EPO can be administer to the subjecttogether with a compound that that decreases the tyrosine phosphataseactivity of a SHP1 in a cell of the nervous system of the subject. Inyet another preferred embodiment, other known neuroprotective agents canbe administered together with a compound that decreases the tyrosinephosphatase activity of a SHP1 in a cell of the nervous system of asubject.

In other general aspects, the invention provides a method for treating anervous system condition related to EPOR in a subject in need thereof,comprising the step of administering to the subject a therapeuticallyeffective dose of a composition that decreases the expression of a SHP1in a cell of the nervous system of a subject. Examples of suchcompositions include for example, compounds that repress SHP1transcription or translation, which can be identified by methodsdescribed infra. In addition, antisense nucleic acids or smallinterfering RNAs (siRNAs) can also be used to reduce the expression ofSHP1 through gene therapy.

The invention is amenable to antisense nucleic acids or siRNA basedstrategies by reducing expression of SHP1 in cells of the nervous systemof a subject. The principle of antisense nucleic acids strategies isbased on the hypothesis that sequence-specific suppression of geneexpression can be achieved by intracellular hybridization between mRNAand a complementary antisense species. The formation of a hybrid RNAduplex may then interfere with the processing/transport/translationand/or stability of the target SHP1 mRNA. Hybridization is required forthe antisense effect to occur. Antisense strategies may use a variety ofapproaches including the use of antisense oligonucleotides, injection ofantisense RNA and transfection of antisense RNA expression vectors.Phenotypic effects induced by antisense effects are based on changes incriteria such as protein levels, protein activity measurement, andtarget mRNA levels.

An antisense nucleic acid can be complementary to an entire codingstrand of a SHP1 gene, or to only a portion thereof. An antisensenucleic acid molecule can also be complementary to all or part of anon-coding region of the coding stand of a SHP1 gene. The non-codingregions (“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequencesthat flank the coding region and are not translated into amino acids.Preferably, the non-coding region is a regulatory region for thetranscription or translation of the SHP1 gene.

An antisense oligonucleotide of the invention can be, for example, alength of about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or morethat is complementary to the nucleotide sequence of human SHP1. Anantisense nucleic acid can be cord using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides that can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,I-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methyleytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. An antisense nucleic acid molecule can be aCC-anomeric nucleic acid molecule. A CC-anomeric nucleic acid moleculeforms specific double-hybrids with complementary RNA in which, thestands run parallel to each other (Gaultier et al. (1987) Nucleic AcidsRes. 15:6625-464 1). The antisense nucleic acid molecule can also a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLet. 215:327-330).

Alternatively, the antisense nucleic acid can also be producedbiologically an expression vector into which a nucleic acid has beensubcloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). That is, a DNA molecule is operably linked toa regulatory sequence in a manner that allows for expression (bytranscription of the DNA molecule) of an RNA molecule that is antisenseto the mRNA encoding a SHP1 protein. Regulatory sequences operablylinked to a nucleic acid cloned in the antisense orientation can bechosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub et al.((1986), Reviews—Trends in Genetics, Vol. 1(1)).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a SHP1 proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix. Antisense nucleic acid molecules can be administered tothe subject via direct injection or surgical implantation in theproximity of the damaged tissues or cells in order to circumvent theirexclusion from the central nervous system (CNS) by an intact blood-brainharrier. Successful delivery of nucleic acid molecules to the CNS bydirect injection or implantation bas been documented (See e.g., Oto etal., (1989), J. Neurosci. Res. 22: 83-91; Goodman & Gilman's ThePharmacological Basis of Therapeutics, 6th ed, pp 244; Williams et al.,(1986), Proc. Natl. Acad. Sci. USA 83: 9231-9235; and Oritz et al.,(1990), Soc. Neurosci. Abs. 386: 18).

Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g. by linking the antisense nucleic acidmolecules to peptides or antibodies that bind to cell surface receptorsor antigens.

The antisense nucleic acid molecules can also be generated in situ byexpression from vectors described herein harboring the antisensesequence. To achieve sufficient intracellular concentrations of theantisense molecules, vector constructs in which the antisense nucleicacid molecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In a preferred embodiment the method of treating a pain in a subject inneed thereof involves the use of small interfering RNA (siRNA). Inseveral organisms, introduction of double-stranded RNA has proven to bea powerful tool to suppress gene expression through a process known asRNA interference. Many organisms possess mechanisms to silence any genewhen double-stranded RNA (dsRNA) corresponding to the gene is present inthe cell. The technique of using dsRNA to reduce the activity of aspecific gene was first developed using the worm C. elegans and has beentermed RNA interference, or RNAi (Fire, et al., (1998), Nature 391:806-811). RNAi has since been found to be useful in many organisms, andrecently has been extended to mammalian cells in culture (see review byMoss, (2001), Curr Biol 11: R772-5).

An important advance was made when RNAi was shown to involve thegeneration of small RNAs of 21-25 nucleotides (Hammond et al., (2000)Nature 404: 293; Zamore et al., (2000) Cell 101: 25-33). These smallinterfering RNAs, or siRNAs, may initially be derived from a lager dsRNAthat begins the process, and are complementary to the target RNA that iseventually degraded. The siRNAs are themselves double-stranded withshort overhangs at each end; they act as guide RNAs, directing a singlecleavage of the target in the region of complementarity (Elbashir etal., (2001) Genes Dev 15: 188-200; Zamore et al., (2000) Cell 101:25-33).

Methods of producing siRNA, 21-23 nucleotides (nt) in length from an invitro system and use of the siRNA to interfere with mRNA of a gene in acell or organism were described in WO0175164 A2, the contents of whichis entirely incorporated herein by reference

The siRNA can also be made in vivo from a mammalian cell using a stableexpression system. For example, a vector system, named pSUPER, that thesynthesis of small interfering RNAs (siRNAs) in mammalian cells, wasrecently reported (Brummelkamp et al., (2002) Science 296: 550-3), andthe contents of which is incorporated herein by reference.

On the pSUPER, the H1-RNA promoter was cloned in front of the genespecific targeting sequence (19-nt sequences from the target transcriptseparated by a short spacer from the reverse complement of the samesequence) and five thymidines (T5) as a termination signal. Theresulting transcript is predicted to fold back on itself to form a19-base pair stem-loop structure, resembling that of C. elegans Let-7.The size of the loop (the short spacer) is preferably 9 bp. A small RNAtranscript lacking a poly-adenosine tail, with a well-defined start oftranscription and a termination signal consisting of five thymidines ina row (T5) was produced. Most importantly, the cleavage of thetranscript at the termination site is after the second uridine yieldinga transcript resembling the ends of synthetic siRNAs, that also containtwo 3′ overhanging T or U nucleotides. The siRNA expressed from pSUPERis able to knock down gene expression as efficiently as the syntheticsiRNA.

The present invention provides a method of treating a nervous systemcondition related to EPOR in a subject in need thereof comprising thesteps of (a) introducing the antisense nucleic acid or siRNA thattargets the mRNA of the SHP1 gene for degradation into the cell ororganism; b) maintaining the cell or orgasm produced (a) underconditions under which the antisense nucleic acid or siRNA interferenceof the mRNA of the SHP1 gene in the cell or organism occurs. The siRNAcan be produced chemically via nucleotide synthesis, from an in vitrosystem similar to that described in WO0175164, or from an in vivo stableexpression vector similar to pSUPER described herein. The siRNA can beadministered similarly as that of the anti-sense nucleic acids describedherein.

The present invention also provides a kit with one or more containerscomprising an inhibitor for SHP1 protein tyrosine phosphatase, and apharmaceutically acceptable diluent.

Methods are known in the art for determining therapeutically andprophylactically effective or active doses for the instantpharmaceutical composition. The “therapeutically effective amount” or“therapeutically active amount” as used herein, means that amount ofactive compound or pharmaceutical agent that elicits the biological ormedicinal response in a tissue system, animal or human at is beingsought by a researcher, veterinarian, medical doctor or other clinician,which includes alleviation of the symptoms of the disease or disorderbeing treated. The term “prophylactically effective amount” or“prophylactically active amount” refers to that amount of activecompound or pharmaceutical agent that inhibits in a subject the onset ofa disorder as being sought by a researcher, veterinarian, medical doctoror of the clinician, the delaying of which disorder is mediated by theactivity of protein tyrosine phosphatases, including but not limited to,SHP1.

Where protein delivery is contemplated, the amount of protein used inthe formulations of the present invention will vary with the biologicalpotency of the protein as well as the desired potency of theformulation, but will generally contain about 1 μg/ml to about 2000μg/ml protein per formulation. Specifically theerythropoietin-containing formulations of the present invention maycontain a “pharmaceutically active amount of erythropoietin”, generallyabout 1000 IU/ml to about 180,000 IU/ml of erythropoietin, wherein120,000 IU is approximately 1000 μg. The erythropoietin may be providedas an aqueous solution of a bulk reagent that is diluted into theformulation of the present invention or may be provided as a driedreagent and reconstituted using the appropriate amount of the aqueousformulation. Dried reagents include, for example, lyophilized orspray-dried erythropoietin. Where erythropoietin is provided as a bulkreagent in formulations of high potency (e.g. greater than 100,000IU/ml), it is preferable that the erythropoietin bulk reagent beprovided in a phosphate buffered solution. This is due to increasedpatient discomfort caused by high concentrations of citric acid bufferstypically used in the preparation of recombinant human erythropoietin.Buffer exchange is achieved using methods well known in the art, such asdiafiltration or dialysis to provide an EPO bulk that contains less than1 millimolar citrate.

The amount of buffering agent useful in the pharmaceutical compositionsof the present invention depends largely on the particular buffer usedand the desired pH of the formulation. The concentration of bufferingions will generally range from about 10 mM to about 30 mM. Suitablebuffer systems to maintain the pH range of about four to Shout nineinclude, but are not limited to, sodium citrate/citric acid, sodiumacetate/acetic acid, sodium or potassium phosphate dibasic/monobasic,and any other pharmaceutically acceptable pH buffering agent(s) known inthe art. The use of a buffer system of sodium phosphate dibasic andsodium phosphate monobasic is preferred. A pH-adjusting agent such as,but not limited to, hydrochloric acid, citric acid, sodium hydroxide, ora salt of any of these, in particular sodium citrate, may be added tothe formulations to adjust the formulation pH to within the desiredformulation pH range. One goal for these formulations is to minimize thepatient discomfort associated with subcutaneous administration of thecitrate-buffered formulations. Therefore phosphate buffer systems areparticularly preferred in all formulations of the present invention,both in the aqueous protein bulk reagent and in the formulation buffercomponent.

One or more ionic tonicity agents may be used in the formulations of thepresent invention. An ionic tonicity agent is any agent capable ofrendering the formulations of the present invention iso-osmotic ornearly iso-osmotic with human blood and carries a positive or negativecharge in aqueous solutions. Typical suitable tonicity agents are wellknown in the art, and include but are not limited to sodium chloride,potassium chloride, ammonium sulfate, glycine, or other amino acids. Thepreferred tonicity agents of the present invention include, but are notlimited to, NaCl, KCl, and glycine, said agent being used at aconcentration in the range of about 0 to about 170 millimolar. Use ofsodium chloride as a tonicity agent is preferred in the formulations ofthe present invention at a concentration of about 75 mM to about 100 mM.The type of tonicity agent and its concentration may influence theproperties of the formulation. In formulations containing more than onetonicity agent the total concentration of tonicity agents is generallyless than 200 mM.

The formulations of the present invention may be prepared by admixingthe formulation reagents in an aqueous solution such that the componentsare mixed substantially uniformly so that none of the components arelocalized. Advantageously all of the formulation components, except theprotein component, can be prepared and adjusted to conditions suitablefor the protein prior to the addition of the protein component.Alternatively, the protein bulk reagent may be diafiltered into anappropriate buffer system, preferably phosphate buffer, and the otherreagents may be added to the protein bulk, and the bulk proteinconcentration can be adjusted appropriately to the desired potency.

The formulations of the present invention are administered to a subjectin need thereof via parental administration including intravenousadministration. Particular routes of parenteral administration include,but are not limited to, intramuscular, subcutaneous, intraperitoneal,intracerebral, intraventricular, intracerebroventricular, intrathecal,intracisternal, intraspinal and/or peri-spinal routes of administrationby delivery via intracranial or intravertebral needles and/or catheterswith or without pump devices. The route of administration may beselected based on the therapeutic indication of the pharmaceuticallyactive protein.

As used for administration of EPO, the phrase “therapeuticallyeffective” or “pharmaceutically active” is generally from about 1 to10000 I.U./kg, preferably from about 50 to 2000 I.U./kg, more preferablyfrom about 50 to 600 I.U./kg, and most preferably from about 50 to 300I.U./kg body weight especially when erythropoietin is administeredsubcutaneously. Advantageously, the formulations of the presentinvention may be administered to a responding subject at any desiredfrequency or time interval between administrations without reducedefficacy. In a preferred dosing regimen, the subject is administered thesustained release formulations of the present invention thrice per twoweeks, once per one per two weeks, once per three weeks, once per month,once per five weeks, once per six weeks, or at more frequent or lessfrequent intervals, or at any combination of frequencies or timeintervals as desired. The effective daily dosing of erythropoietin (EPO)is preferably from about 4000 to about 9000 I.U. (equivalent to about60,000 I.U. to about 120,000 I.U. every two weeks). Most preferably theeffective daily dosing of erythropoietin (EPO) is 5715 I.U. (equivalentto About 80,000 I.U. every two weeks). A preferred dosing may be onceper three weeks, particularly for subjects receiving chemotherapy forthe treatment of cancer, since many chemotherapeutic regimens areadminstered on a once per three-week schedule. However, any dosingschedule of a therapeutic protein, such as EPO, formulated according tothe present invention, can be easily coordinated with regular visits tothe treating physician or with the dosing schedule of another agent,such as an anti-tumor agent, as is desirable for the patient. Thisallows the EPO regimen and the chemotherapeutic regimen to beadministered simultaneously or in parallel, providing an economic anddesirable benefit for the subject. EPO administration is delayed orwithheld if the patient, male or female, exhibits a hemoglobin level inexcess of about 18 g/dL for a human male and about 16 g/dL for a humanfemale.

For therapeutic purposes, the term “jointly active amount” as usedherein, means that amount of each active compound or pharmaceuticalagent, alone or in combination, that elicits the desired biological ormedicinal response in a tissue system, animal or human that is beingsought by a researcher, veterinarian, medical doctor or other clinician,which includes alleviation of the symptoms of the disease or disorderbeing treated. Particularly, the biological or medicinal response isinhibition of dephosphorylation of the EPOR, or prolongation orenhancing of phosphorylation of the EPOR.

The compounds of the present invention may also be present in the formof a pharmaceutically acceptable salt or salts. For use in medicine, thesalt or salts of the compounds of is invention refer to non-toxic“pharmaceutically acceptable salt or salts.” Other salts may, however,be useful in the preparation of compounds according to this invention orof their pharmeceutically acceptble salts. Representative organic orinorganic acids include, but am not limit to, hydrochloric, hydrobromic,hydriodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic,glycolic, lactic, succinic, maleic, fumaric, malic, tartaric, citric,benzoic, mandelic, methanesulfonic, hydroxyethanesulfonic,benezenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic,p-toluenesulfonic, cyclohexanesulfamic, salicylic, saccharinic ortrifluoroacetic acid. Representative basic/cationic salts include, butare limited to, benzathine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine, procaine, aluminum, calcium, lithium,magnesium, potassium, sodium or zinc.

Method of Identifying a Compound Useful for Treating a Nervous SystemCondition Related to EPOR

The invention further provides efficient methods of identifyingcompounds that are useful for treating a nervous system conditionrelated to EPOR. Generally, the methods involve identifying compoundsthat decease: 1) the expression of a SHP1 protein; or 2) the tyrosinephosphatase activity of a SHP1 protein.

The compound identification methods can be in conventional laboratoryformat or for high throughput. The term “high throughput” refers to anassay design that allows easy screening of multiple samplessimultaneously, and capacity for robotic manipulation. Another desiredfeature of high throughput assays is an assay design that is optimizedto reduce reagent usage, or minimize the number of manipulations inorder to achieve the analysis desired. Examples of assay formats include96-well or 384-well plates, levitating droplets, and “lab on a chip”microchannel chips used for liquid handling experiments. It is wellknown by those in the art that as miniaturization of plastic molds andliquid handling devices are advanced, or as improved assay devices aredesigned, greater numbers of samples may be performed using the designof the present invention.

Candidate compounds encompass numerous chemical classes althoughtypically they are organic compounds. Preferably, they are small organiccompounds, i.e., those having a molecular weight of more than 50 yetless than about 2500. Candidate compounds comprise functional chemicalgroups necessary for structural interactions with polypeptides, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups andmore preferably at least three of the functional chemical groups. Thecandidate compounds can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate compounds also canbe biomolecules such as peptides, saccharides fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like. Where the compound is anucleic acid, the compound typically is a DNA or RNA molecule, althoughmodified nucleic acids having non-natural bonds or subunits are alsocontemplated.

Candidate compounds am obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and direct synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptide and the like. Candidate compoundscan also be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries: synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection (Lam (1997) Anticancer Drug Des.12:145). Alternatively, libraries of nail compounds in the form ofbacterial, fungal, plant and animal extracts are available or readilyproduced. Additionally, natural and synthetically produced libraries andcompounds can be readily modified through conventional chemical,physical, and biochemical means.

Further, known pharmacological agents may be subjected to dire or randomchemical modifications such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs of the agents.Candidate compounds can be selected randomly or can be based on existingcompounds that bind to and/or modulate the tyrosine phosphatase activityof SHP1. Therefore a source of candidate agents is libraries ofmolecules based on the known SHP1 inhibitors, in which the structure ofthe compound is changed at one or more positions of the molecule tocontain more or fewer chemical moieties or different chemical moieties.The structural changes made to the molecules in creating the librariesof analog activators/inhibitors can be directed, random, or acombination of both directed and random substitutions and/or additions.One of ordinary skill in the art in the preparation of combinatoriallibraries can readily prepare such libraries based on the existing SHP1inhibitors.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. that may be used to facilitate optimaprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

1. Identify Compounds that Decrease SHP1 Expression.

As used herein, “compounds that decreases the SHP1 protein expression”include compounds that decrease SHP1 gene transcription and/ortranslation. The invention provides a method of identifying such acompound, which comprises the steps of contacting a compound with aregulatory sequence of the SHP1 gene or a cellular component thatsubsequently binds to the regulatory sequence; and determining theeffect of the compound on the expression of a gene controlled by theregulatory sequence; wherein the regulatory sequence of the SHP1 gene iseither within a host cell or in a cell-free system. The term “regulatorysequence” is as defined supra.

In a preferred embodiment, the method involves a regulatory sequence ofthe SHP1 gene within a host cell, preferably a cell of the nervoussystem. The cell-based assay comprises the step of: (1) contacting acompound with a cell having a regulatory sequence for a SHP1 gene or acellular component that binds to the regulatory sequence for a SHP1gene; (2) measuring the effect of the compound on the expression of aSHP1 or a reporter gene controlled by the regulator sequence; and (3)comparing the effect of the compound with that of a reference control.

The host cell can be a native SHP1 host cell, or a recombinant hostcell. The reference control contains only the vehicle in which thetesting compound is dissolved. Several assay methods can be used tomeasure the effect of the compound on the expression of the SHP1 orreporter gene inside a cell. For example, gene or protein fusionscomprising the regulatory sequence for a SHP1 linked to a reporter genecan be used. As used rein, “a reporter gene” refers to a gene encoding agene product which can be measured using conventional lab techniques.Such reporter genes include but are not limited to genes encoding greenfluorescent protein (GFP), β-galactosidase, luciferase, chloramphenicolacetyltransferase, β-glucuronidase, neomycin phosphotransferase, andguanine xanthine phosphoribosyltransferase. The gene fusion isconstructed such that only the transcription of the reporter gene isunder control of the SHP1 regulatory sequence. The protein fission isconstrue so that both the transcription and translation of the reportergene protein are under control of the SHP1 regulatory sequence.Preferably, a second gene or protein fusion comprising the same reportergene but a different regulatory sequence (i.e., a regulatory sequencefor a gene unrelated to SHP1 family) can be used to increase thespecificity of the assay.

The effect of the compound on the expression of the reporter gene, suchas GFP, can be measured by methods known to those skilled in the art.For example, the effect of the compound on expression of GFP can bemeasured as the effect of the compound on emissions of greenfluorescence from the cell using a fluorometer. Alternatively, acellular phenotype attributed to a SHP1, such as the phosphorylationlevel of Jak2 or EPOR, can also be used to measure the effect of thecompound on the expression of the SHP1 protein. In addition, the effectof the compound can be assayed by measuring the amount of SUP orreporter mRNA or protein inside the cell directly using methods such asNorthern Blot, RT-PCR, SDS-PAGE, Western Blot, etc., which are known tothose skill in the art.

Note that the cell-based method described supra not only identifiescompounds that regulate SHP1 expression directly via binding to theregulatory sequence of a SHP1 gene, but also identifies compounds thatregulate SHP1 expression indirectly via binding to other cellularcomponents whose activities influence the SHP1 expression. For example,compounds that modulate the activity of a transcriptional activator orinhibitor for SHP1 genes can be identified using the method describedherein.

In another embodiment, the method involves a regulatory sequence of theSHP1 gene in a cell-free assay system. The cell-free assay comprises thestep of: (1) contacting a compound to the regulatory sequence for a SHP1gene or a cellular component that binds to the regulatory sequence for aSHP1 gene in a cell-free assay system; (2) measuring the effect of thecompound on the expression of the SHP1 or reporter gene controlled bythe regulatory sequence; and (3) comparing the effect of the compoundwith that of a reference control. The reference control contains onlythe vehicle in which the testing compound is dissolved. Examples of thecell-free assay system include the in vitro translation and/ortranscription system, which are known to those skilled in the art. Forexample, the full length SHP1 cDNA, including the regulatory sequence,can be cloned into a plasmid. Then, using this construct as thetemplate, SHP1 protein can be produced in an in vitro transcription andtranslation system. Alternatively, synthetic SHP1 mRNA or mRNA isolatedfrom SHP1 protein producing cells can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts. The effect of the compound on theexpression of the SHP1 or reporter gene controlled by the regulatorysequence can be monitored by direct measurement of the quantity of SHP1or reporter mRNA or protein using methods described supra.

2. Identify an Inhibitor for a SHP1 Tyrosine Phosphatase Activity

An “inhibitor” for a SHP1 tyrosine phosphate activity refers to aninhibitory molecule identified using in vitro and in vivo assays forSHP1 tyrosine phosphatase activity. In particular, “inhibitors”, referto compounds that decrease, block, prevent, delay activation,inactivate, desensitize or down regulate SHP1 tyrosine phosphataseactivity, or speed or enhance deactivation of the SHP1 tyrosinephosphatase activity.

The invention further provides a method of identifying an inhibitor of aSHP1 tyrosine phosphatase activity, comprising the steps of:

1) contacting a test compound with a SHP1 protein or an active fragmentthereof; and

2) determining the ability of the test compound to decrease the tyrosinephosphatase activity of SHP1.

The amount of time necessary for contacting the test compound with theSHP1 protein is empirically determined, for example, by running a timecourse with a known SHP1 inhibitor, and measuring the tyrosinephosphatase activity of SHP1 as a function of time.

A variety of assay methods can be used to determine the effect of thecompound on the tyrosine phosphatase activity of SHP1. Some of the assaymethods are disclosed in WO99/54450. For example, SHP1 phosphotaseactivity can be measured as a function of the ability of SHP1 todephosphorylate a substrate, such as a phosphorylated JAK2. Inparticular, a phosphorylated peptide derived from JAK2. Thedephosphorylation reaction can be monitored using a malachite greenassay (Lanzetta et al., Anal Biochem. 1979, 100:95-7), which measuresthe release of inorganic phosphate from the substrate. Alternatively,SHP1 phosphotase activity can be mead in vivo by changes in cellularphysiology affected by the SHP1 phosphotase activity, such as theinactivation of the EPOR signal transduction pathway.

Alternatively, binding assays can be used to identify to a compound thatbinds to a SHP1 protein, and potentially is capable of inhibiting thephosphatase activity of SHP1. One exemplary method of such a bindingassay comprises the steps of:

-   -   1) combining a test compound, a labeled ligand for a SHP1 pin,        and a SHP1 protein or an active fragment of; and    -   2) measuring the binding of the test compound to the SHP1        protein or active fragment thereof by a reduction in the amount        of labeled ligand binding to the SHP1 protein or active fragment        thereof.

The amount of labeled ligand binding to the SHP1 protein or activefragment thereof can be measured by separating the SHP1 from unboundlabeled ligand. The separation can be accomplished in a variety of ways.Conveniently, at least one of the components is immobilized on a solidsubstrate, from which the unbound components may be easily separated.The solid substrate can be made of a wide variety of materials and in awide variety of shapes, e.g., microtiter plate, microbead, dipstick,resin particle, etc. The substrate preferably is chosen to maximizesignal to noise ratios, primarily to minimize background binding, aswell as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, or rinsing a bead, particle, chromatographic column orfilter with a wash solution or solvent. The separation step preferablyincludes multiple rinses or washes. For example, when the solidsubstrate is a microtiter plate, the wells may be washed several timeswith a washing solution, that typically includes those components of theincubation mixture that do not participate in specific bindings such assalts, buffer, detergent, non-specific protein, etc. Where the solidsubstrate is a magnetic bead, the beads may be washed one or more timeswith a washing solution and isolated using a magnet.

The ligand for SHP1 can be a polypeptide that binds specifically toSHP1, such as an antibody for SHP1, or chemical compound that bindsspecifically to SHP1. A wide variety of labels can be used to label theSHP1 ligand, such as those that provide direct detection (e.g.,radioactivity, luminescence, optical or electron density, etc), orindirect detection (e.g., epitope tag such as the FLAG epitope, enzymetag such as horseradish peroxidase, etc.).

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

The SHP1 protein or active fragment thereof can be substantiallypurified, or expressed from a host cell. The term “cell” refers to atleast one cell, but includes a plurality of cells appropriate for thesensitivity of the detection method. Cells suitable for the presentinvention may be a cell of the nervous system that expresses SHP1endogenously, or a recombinant cell, such as a bacterial, yeast, oreukaryotic cell, that expresses SHP1 recombinantly.

In preferred embodiments of the invention the methods of identifying acompound for treating a nervous system condition related to EPOR, fibercomprise the steps of

-   -   a) contacting a neuronal cell with the test compound;    -   b) inducing neurotoxicity in the neuronal cell;    -   c) assaying the cell survival rate in the presence of the test        compound,    -   and comparing the cell survival rate with that of a control        wherein the neuronal cell is not treated with the test compound.

Neurotoxicity in the neuronal cells can be induced by a variety ofmethods known to those skilled in the art. For example, neurotoxicity ina primary hippocampal neuron. can be induced by challenging thehippocamapal neurons with glutamate. Such methods are known in the art.

The survival rate of the neuronal cells can be measured by a variety ofmethods known to those skilled in the art. For example, the survivalrate of the neuronal cells can be measured using trypan blue exclusionassay.

Compounds of Formula I, II, and III were identified as inhibitors forSHP1 tyrosine phosphatase activity using methods of the invention.

inhibited SHP1 phosphatase activity with an IC₅₀ of 22 μM under theassay conditions. Studies related this compound have been describedpreviously (Safarik et al., Spectroscopy Letters (1999), 32(3), 443-462;Malesevic et al., Heterocycles (1995), 41(12), 2691-9; Karminski-Zamolaet al., Heterocycles (1994), 38(4), 759-67).

inhibited SHP1 phosphatase activity with an IC₅₀ of 25.7 μM under theassay conditions. Studies related to this compound have been describedpreviously (for example in JP 92-129774).

inhibited SHP1 phosphatase activity with an IC₅₀ of 4.3 μM under theassay conditions. The following examples illustrate the presentinvention without, however, limiting the same thereto.

EXAMPLE 1 Isolation and Culturing of Primary Cells

A Rat was sacrificed at eighteen days post-coitus (d.p.c.). Rat embryoswere isolated and the brains dissected in ice cold Hanks Balanced SaltSolution (HBSS). The meninges were carefully dissected away and theouter layer of frontal cortex removed and placed in 0.05 trypsin EDTAfor twenty minutes. The trypsin was then removed and the tissuetriturated in plating media (DMEM w/10% FBS, 1%Penicillin/streptomycin). The dissociated cells were then plated on theappropriate substrate coated culture dishes in plating media.

Primary cortical neurons were cultured on poly-d-lysine coated cultureware (BIOCOAT from Beckton Dickson) for twenty-four hours in platingmedia then transferred to Neurobasal media (LTI) containing 1×B-27 serumfree supplement (LTI) and 1% Penicillin/streptomycin. After three daysin the culture, cells were treated with 1 μM Cytosineβ-D-Ambino-Furanoside (ARAC) to further select for neurons overnon-neuronal cells. The neurons were culture in the ARAC containingmedia for three additional days (seven days total in culture) beforebeing used for experiments. On the day of the experiment, cultures werechanged to B-27 free media for six hours. EPO (10 pM) was then added tothe cultures for the appropriate time interval.

UT-7 cells were obtained from the New York Blood Center and culturedaccording to [12]. Briefly UT-7 cells were grown in Iscove's media (LifeTechnologies) containing 10% fetal bovine serum (FBS), 1%Penicillin/streptomycin, and 1 U/ml rhEPO. EPO was removed from thecultures twenty-four hours prior to the experiment. On the day of theexperiment, the cells were changed to Iscove's serum free media for fourhours, and then stimulated with EPO (2.5 nm or 0.25 nm) for theappropriate time interval.

Primary Astrocytes were isolated as described above and plated onun-coated tissue culture plastic dishes in plating media. Cultures ofprimary rat astrocytes were obtained by leaving the cells in platingmedia for fourteen days, changing the media every four days. At the endof fourteen days, the cells were used for analysis.

EXAMPLE 2 Lysate Preparation and Western Analysis

The precise control of signaling through receptor pathways requires theproper control of activation and inactivation events to initiate andterminate signaling. Activation events such as changes in receptorconformation or phosphorylation state are balanced by termination eventssuch as receptor internalization, a return to basal conformation,enzymatic processes that return the receptor to its basalphosphorylation state or some combination of these events. Someligand-receptor systems make use of feedback mechanisms to regulatetheir own activity [19] thereby providing an additional level of controlover their signaling activity.

According to the present invention, 1) EPO causes the phosphorylation ofthe EPO receptor and this phosphorylation is sustained relative tohematopoietic cells, 2) the phosphorylation of the downstream targets ofEPO signaling ERK1/ERK2 is also sustained and 3) the expression of thenegative regulator SHP1 is deceased following treatment with EPO. Theobserved changes in expression, and thereby the activity of SHP1 as aresult of EPO can be responsible, at least in part for one of the uniqueproperties of EPO, namely its increased potency, in neurons.

Cells were lysed with lysis buffer (20 mM Hepes, 79, 10 mM KCl, 0.1 mMNaVO₄, 1 mM EDTA, 1 mM EGTA, 0.2% NPA-40, 10% Glycerol, 0.2 mM PMSF) forten minutes on ice Extracts were spun at 15,000 rpm for two minutes andthe supernatants were subjected to SDS-PAGE. Samples were transferred tonitrocellulose membrane (IMMOBILON-P, Millipore) and blocked with TBS-T(20 mM Tris-Cl, pH 7.6, 150 mM NaCl, 0.1% Tween-20) containing 1% FBS.Membranes were subsequently probed with antibodies against SHP-1 (mousemonoclonal from Transduction Laboraties) or Erk1/2 (p44/42) (rabbitpolyclonal from Cell Signaling). A secondary antibody, conjugated tohorseradish peroxidase was used to detect the proteins and theimmunoreactive bands were visualized using a chemiluminescence kit(Amersham Pharmacia Biotech, Piscataway, N.J.).

Cell lysis and immunoprecipitation was carried out as previouslydescribed [16]. Briefly, cells were lysed with lysis buffer (10 mMTris-Cl, pH 9.0, 150 mM NaCl, 5 mM EDTA, 10% Glyrcerol, 1 mM sodiumvanadate, 1% Igepal CA-630, protease/phosphatase inhibitor cocktailsfrom Sigma). After fifteen minutes on ice, the extracts were centrifugedfor ten minutes at 15,000 rpm and the supernatants were used forimmunoprecipitation. Anti-EPO receptor antibody (Maine Biotech) wasincubated with the cell extracts for one and one-half hours at 4° C. andthen rocked for one hour with G-sepharose beads. After one hour, themixture was washed twice with lysis buffer and once with lysis buffercontaining 0.1% Igepal CA-630 (NP40). The samples were subsequentlyboiled in Laemlli sample buffer and subjected to SDS-PAGE andimmunoblotting.

EPO treatment results in sustained activation of the MAP Kinase pathwayas seen by phosphorylation of ERK1/ERK2. Primary cortical neurons werecultured for seven days in Neurobasal media containing B-27 serum freesupplement. The cells were changed to B-27 free media for six hoursprior to the experiment to decrease basal phosphorylation of ERK1/ERK2.EPO was added to the neurons at a concentration of 10 pm. Preparation oflysates and subsequent analysis with SDS-PAGE showed that thephosphorylation of ERK1/ERK2 was detectable at very low levels by 1 hrand increased up to six hours (FIG. 1). The phosphorylation reached amaximum at six hours then deceased to near basal levels by twenty-fourhours. In contrast, the phosphorylation of ERK1/ERK2 in UT-7 cells inresponse to EPO (2.5 nm, 0.25 nm) was detectable by five minutes andreached a maximum at fifteen minutes. By thirty minutes, thephosphorylation of ERK1/2 had diminished to near undetectable levels(FIG. 1).

Previous reports have implicated the ERK1/ERK2 pathway inneuroprotection. Glutamate has been reported to increase thephosphorylation of ERK1/ERK2 [29] and inhibits of the ERK1/ERK2 kinasepathway have been shown to be neuroprotective [23, 2, 25]. Thus thesustained activation of the ERK1/ERK2 pathway by EPO seems to contradictits role as a neuroprotectant. However, the activation of ERK1/ERK2 maynot be as important for EPO mediated neuroprotection as the eventualinactivation of this pathway. According to the present invention, thephosphorylation of ERK1/ERK2 was terminated by twenty-four foursfollowing EPO treatment; the phosphorylation of ERK1/ERK2 began todecrease even thou the EPO receptor was still phosphorylated. Thedecrease in ERK1/ERK2 is most likely due to a mechanism different thanthe inactivation of the EPOR signal and involves a separate process suchas depletion of ERK1/ERK2 or a process that might or might not bemediated by EPO directly.

EPO treatment results in tee sustained activation/phosphorylation of theEPO rector in primary neurons. Primary cortical neurons and UT-7 cellswere treated with EPO and immunoprecipitated with the EPOR antibody. Thepreparations were then subjected to SDS-PAGE and the phosphorylated EPORwas detected using an anti-p-tyrosine antibody. Treatment of corticalneurons with EPO (10 pm) resulted in an increased phosphorylation of theEPOR that was detectable by one hour and persisted to six hours aftertreatment (FIG. 2). In contrast, treatment of UT-7 cells with EPO (2.5nm) resulted in a rapid, transient phosphorylation of the EPOR that wasdetectable by two minutes, peaked at ten minutes, and returned to nearbasal levels by thirty minutes (FIG. 2).

EPO decreases the expression of SHP1 protein in primary corticalneurons. Since there is a decrease in SHP1 mRNA expression in corticalneurons, we sought to determine if that translated into a decrease inprotein levels. Primary cortical neurons and UT-7 cells were treatedwith 10 pm EPO and 2.5 nM EPO respectively. Cells were lysed, subjectedto SDS-PAGE, and Western blots were probed with an antibody to SHP1. Aswith mRNA expression, SHP1 protein in primary cortical neurons is seento decrease over the 6 hour interval (FIG. 5). In UT-7 cells, additionof EPO did not change SHP1 protein levels which remained constantthrough 1 hour (FIG. 5).

EXAMPLE 3 RNA Isolation and RT-PCR

Primary cortical neurons and UT-7 cells were cultured as describedabove. Following EPO treatment total RNA was isolated using the RNAeasykit (Qiagen). RNA was then quantitated and 2.5 μg was used to make firststrand cDNA using the Superscript™ Preamplification System (GibeoBRL).PCR was hen carried out on the first stand cDNA using oligonucleotideprimers designed against human rat shp-1. To control for cDNA content,GAPDH was amplified simultaneously. The target molecules were amplifiedfollowing oligonucleotide primers: human SHP-1 (5′-ttcctggaccagatcaaccag(SEQ ID NO:6) and 3′-cttcctcttgagggaccacttgc SEQ ID NO:1), Rat SHP-1(5′-aaaggccggaacaatgtgt (SEQ ID NO:2) and 3′-ggatgg tcttctggatgtca (SEQID NO:3)), and Rat GAPDH (5′-ggagtctactggcgtcttcac (SEQ ID NO:4) and3′-aaggccatgccagtgagcttc (SEQ ID NO:5)).

EPO induces a dose dependant decreased the expression of SHP1 mRNA inprimary neurons and in primary astrocytes. To determine a possiblemechanism for the sustained phosphorylation of EPO targets in neuronsthe expression of SHP1, a negative regulator of EPO receptorphosphorylation was examined. Primary Neurons and astrocytes cultured asdescribed above were treated with EPO (100 fm, 10 pm, 1 nm). Total RNAwas isolated and then subjected to RT-PCR, using primers for the RatSHP1 to determine SHP1 mRNA expression levels. RAT GAPDH served as acontrol. Twenty-four hours of pre-treatment of EPO at 10 pm or 1 nmresulted in a decrease in the expression of SHP1 mRNA in primarycortical neurons FIG. 3). EPO at 100 fm did not alter the expressionlevel of SHP1. Similarly, in astrocytes, a dramatic decrease in SHP1mRNA is seen in 10 pM and 1 nM EPO treated cells. 100 fM EPO does notaffect expression levels (FIG. 6). In UT-7 cells, three hours ofpre-treatment of EPO (2.5 nm) resulted in an increase of SHP1 mRNA.However, in comparison with the three hour pre-treatment, cellspre-treated with EPO for six or twenty-four hours did not show anydifference.

The potential implications of EPO's attenuation of SHP1 expression inthe CNS can be significant. EPO's increased potency in neurons, combinedwith the sustained activation of its signal can be important consideringthe relatively low concentrations of EPO that are present in the CNS [8]and the low amounts that can cross the blood brain barrier and enter theCNS from the systemic circulation. Enhancing the signal can be a way bywhich the brain can best make use of the EPO that is available to it.

SHP1 has also been suggested to function as a positive signalingmolecule in non-neuronal cells of the nervous system. Astrocytes andmicroglia have been reported to rely on SHP1 for activation. Previousreports have demonstrated that astrocytes and microglia express EPORsuggesting that these cell types can be responsive to EPO [24].According to the present invention, EPO decreases the expression of SHP1in astrocytes in a dose dependant manner. The activation of astrocytesand microglia has been implicated in damage to the CNS associated with anumber of pathological conditions including bacterial infection [9],Alzheimers disease [17], and focal brain injury [32, 15, 31]. Themechanism by which astrocytes and microglia contribute to neural damageremains unclear but may be due in part to their release of cytokinesfollowing activation and their contribution to the inflammatory responsein the injured brain. The ability of EPO to decrease SHP1 expression canattenuate the positive signal that leads to the activation of thesenon-neuronal cells, thereby preventing the damage that is associatedwith them. The observation that EPO has a similar effect on theexpression of SHP1 in astrocytes provides evidence that EPOs actions inthe CNS are not limited to neuronal cells.

A decrease in SHP1 mRNA expression is detectable in cortical neuronsfollowing three hour exposure to EPO. The expression of SHP1 mRNA wasexamined in primary cortical neurons and UT-7 cells treated with EPO (10pm) for tree, six or twenty-four hours to determine the time to induce achange in SHP1 mRNA expression. In primary neurons, a decrease in SHP1mRNA expression was detectable after three hours of exposure to EPO.SHP1 expression continued to decrease at six hours and was virtuallyundetectable by twenty-four hours (FIG. 4). EPO did not cause a decreasein SHP1 expression in UT-7 cells. In fifty the amounts of SHP1 mRNAseemed to increase by three hours and remain elevated out to twenty-fourhours FIG. 4).

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1. A method of identifying a compound useful for treating a nervoussystem condition related to EPOR, comprising the steps of: 1) contactinga test compound with a SHP1 protein or an active fragment thereof and 2)determining the ability of the test compound to decrease the tyrosinephosphatase activity of SHP
 1. 2. The method of claim 1, furthercomprising the steps of a) contacting a neuronal cell with the testcompound; b) inducing neurotoxicity in the neuronal cell; c) assayingthe cell survival rate in the presence of the test compound, andcomparing the cell survival rate with that of a control, wherein theneuronal cell is not treated with the test compound.
 3. The method ofclaim 1, wherein said SHP1 protein or active fragment thereof issubstantially purified.
 4. The method of claim 1, wherein SHP1 proteinor active fragment thereof is expressed from a host cell.
 5. A method ofidentifying a compound useful for treating a nervous system conditionrelated to EPOR, comprising the steps of: c) contacting a test compoundwith a regulatory sequence for a SHP1 gene or a cellular component thatbinds to the regulatory sequence for a SHP1 gene; and d) determiningwhether the test compound decreases the expression of a gene controlledby said regulatory sequence.
 6. The method of claim 5, furthercomprising the steps of a) contacting a neuronal cell with the testcompound; b) inducing neurotoxicity in the neuronal cell; c) assayingthe cell survival rate in the presence of the test compound, andcomparing the cell survival rate with that of a control, wherein theneuronal cell is not treated with the test compound.
 7. The method ofclaim 5, wherein the gene controlled by the SHP1 regulatory sequence isa reporter gene.
 8. The method of claim 5, wherein the gene controlledby the SHP1 regulatory sequence is an SHP1 gene.
 9. The method ofidentifying a compound useful for treating a nervous system conditionrelated to EPOR, comprising the steps of: e) combining a test compound,a labeled ligand for a SHP1 protein, and a SHP1 protein or an activefragment thereof; and f) measuring the binding of the test compound tothe SHP1 protein or active fragment thereof by a reduction in the amountof labeled ligand binding to the SHP1 protein or active fragmentthereof.
 10. The method of claim 9, further comprising the steps of a)contacting a neuronal cell with the test compound; b) inducingneurotoxicity in the neuronal cell; c) assaying the cell survival ratein the presence of the test compound, and comparing the cell survivalrate with that of a control, wherein the neuronal cell is not treatedwith the test compound.